Diabetes-associated endothelial injury and vascular dysfunction is related to local, free radical-related processes. Pharmacological modulation of this endothelial cell death and injury may provide novel avenues for the experimental therapy of the vascular dysfunction in advanced diabetes. In the prior funding period, we obtained data in cultured endothelial cells showing that oxidants produced in the Vicinity of endothelial cells during hyperglycemia trigger an intracellular cascade culminating governed by the nuclear enzyme poly (ADP-ribose) polymerase (PARP). This results in activation of PARP, which initiates an energy consuming metabolic cycle, with resultant cell dysfunction. Our data also show that high glucose activates the PARP pathway in endothelial cells in vitro. Pharmacological inhibition or genetic inactivation of PARP reduces the development of oxidant- or high glucose mediated endothelial injury. Destruction of functional islet cells with streptozotocin in rats induced chronic hyperglycemia, extravascular production of oxidants, activation of PARP in the endothelium, and a loss of endothelial function. PARP inhibition, starting after the time of islet destruction, maintained and restored normal vascular responsiveness, despite the persistence of severe hyperglycemia. Now we propose mechanistic studies and additional, definitive in vivo studies to establish the role of PARP in diabetic vascular failure. We will establish the molecular triggers leading to PARP activation in diabetes. In addition, we will characterize changes in sentinel markers of endothelial injury, and the role of the PARP pathway. The in vivo studies will be complemented by in vitro studies investigating the mode of high glucose induced PARP activation and cell death in macro- and microvascular endothelial cells. A further aim is to perform studies in chronic rat diabetes models to delineate the role of PARP in the development of retinopathy, nephropathy and neuropathy associated with diabetes mellitus. We will also extend the studies from the vascular dysfunction to the myocardial dysfunction (diabetic cardiomyopathy). The final aim of the study is to investigate the activation of PARP in human microvasculature, and to correlate the degree of PARP activation with the degree of endothelial dysfunction in humans.